Cuticular Wax Biosynthesis Research Articles

Genome and transcriptome analyses reveal genes involved in the formation of fine ridges on petal epidermal cells in Hibiscus trionum.

Hibiscus trionum, commonly known as the 'Flower of an Hour', is an easily cultivated plant in the Malvaceae family that is widespread in tropical and temperate regions, including drylands. The purple base part of its petal exhibits structural colour due to the fine ridges on the epidermal cell surface, and the molecular mechanism of ridge formation has been actively investigated. We performed genome sequencing of H. trionum using a long-read sequencing technology with transcriptome and pathway analyses to identify candidate genes for fine structure formation. The ortholog of AtSHINE1, which is involved in the biosynthesis of cuticular wax in Arabidopsis thaliana, was significantly overexpressed in the iridescent tissue. In addition, orthologs of AtCUS2 and AtCYP77A, which contribute to cutin synthesis, were also overexpressed. Our results provide important insights into the formation of fine ridges on epidermal cells in plants using H. trionum as a model.

A 3-Ketoacyl-CoA Synthase 10 (KCS10) Homologue from Alfalfa Enhances Drought Tolerance by Regulating Cuticular Wax Biosynthesis.

Cuticular wax, forming the first line of defense against adverse environmental stresses, comprises very long-chain fatty acids (VLCFAs) and their derivatives. 3-Ketoacyl-CoA synthase (KCS) is a rate-limiting enzyme for VLCFA biosynthesis. In this study, we isolated KCS10, a KCS gene from alfalfa, and analyzed the effect of gene expression on wax production and drought stress in transgenic plants. MsKCS10 overexpression increased compact platelet-like crystal deposition and promoted primary alcohol biosynthesis through acyl reduction pathways in alfalfa leaves. Overexpression of MsKCS10 induced the formation of coiled-rodlet-like crystals and increased n-alkane content through decarbonylation pathways in tobacco and tomato fruits. Overexpression of MsKCS10 enhanced drought tolerance by limiting nonstomatal water loss, improving photosynthesis, and maintaining osmotic potential under drought stress in transgenic tobacco. In summary, MsKCS10 plays an important role in wax biosynthesis, wax crystal morphology, and drought tolerance, although the mechanisms are different among the plant species. MsKCS10 can be targeted in future breeding programs to improve drought tolerance in plants.

Formation of plant resistance to pathogens involving epigenetic factors and phytohormones

The development of resistance to plant pathogens is determined by the presence of resistance genes and regulation of their activity due to phytohormones, the activity of the nuclear pore complex, and epigenetic modifications at the post-transcriptional and post-translational levels of DNA and histones, respectively. Changes in gene expression due to such modifications can be inherited in generations and contribute to the selection of resistant plants in populations.The nuclear pore complex is composed of nucleoporins, a nuclear pore basket, cytoplasmic filaments and is able to selectively transport transcription factors to the nucleus from the cytoplasm and mRNA in the opposite direction, affecting gene expression. Methylation is the most common and well-studied among the epigenetic modifications. It is described for DNA and histones and ensures genome stability and the availability of transcription factors. Incorporation of methyl groups to amino acid residues is not always a factor in gene silencing for histones. The number of the incorporated groups as well as an amino acid they are attached to are crucial. Histone acetylation is associated with the transition to transcriptional activity.The general control histone acetyltransferase TaGCN5 is able to promote expression of the enoyl-CoA reductase gene in allohexaploid wheat, which is involved in the biosynthesis of cuticular wax as one of the resistance factors. The reverse process of deacetylation acts both as a positive regulator, through ethylene response factors, and a negative one, as it interferes with acetylation and methylation of histone amino acids. Jasmonic and salicylic acids, along with combinations of the mentioned phytohormones, mediate formation of systemic acquired resistance in addition to ethylene. Auxin is a positive phytohormone for pathogens due to its ability to influence the structure of the cell wall. At the same time, pathogens, due to effectors, are able to inhibit the plant’s immune responses, so there is a constant “arms race” resulting in the selection of more effective means of penetration and development in the plant as well as its protective reactions. Investigation of the mechanisms of resistance formation and identifying the main factors of resistance, such as either presence of a gene sequence and/or factors regulating its expression, are of great importance.

Comprehensive analysis of KCS gene family in pear reveals the involvement of PbrKCSs in cuticular wax and suberin synthesis and pear fruit skin formation.

Cuticular wax, cutin and suberin polyesters covering the surface of some fleshy fruit are tightly associated with skin color and appearance.β-Ketoacyl-CoA synthase (KCS) is a rate-limiting enzyme participating in the synthesis of very-long-chain fatty acids (VLCFAs), the essential precursors of cuticular waxes and aliphatic monomers of suberin. However, information on the KCS gene family in pear genome and the specific members involved in pear fruit skin formation remain unclear. In the present study, we performed an investigation of the composition and amount of cuticular waxes, cutin and aliphatic suberin in skins of four sand pear varieties with distinct colors (russet, semi-russet, and green) and demonstrated that the metabolic shifts of cuticular waxes and suberin leading to the significant differences of sand pear skin color. A genome-wide identification of KCS genes from the pear genome was conducted and 35 KCS coding genes were characterized and analyzed. Expression profile analysis revealed that the KCS genes had diverse expression patterns among different pear skins and the transcript abundance of PbrKCS15, PbrKCS19, PbrKCS24, and PbrKCS28 were consistent with the accumulation of cuticular waxes and suberin in fruit skin respectively. Subcellular localization analysis demonstrated that PbrKCS15, PbrKCS19, PbrKCS24 and PbrKCS28 located on the endoplasmic reticulum (ER). Further, transient over-expression of PbrKCS15, PbrKCS19, and PbrKCS24 in pear fruit skins significantly increased cuticular wax accumulation, whereas PbrKCS28 notably induced suberin deposition. In conclusion, pear fruit skin color and appearance are controlled in a coordinated way by the deposition of the cuticular waxes and suberin. PbrKCS15, PbrKCS19, and PbrKCS24 are involved in cuticular wax biosynthesis, and PbrKCS28 is involved in suberin biosynthesis, which play essential roles in pear fruit skin formation. Moreover, this work provides a foundation for further understanding the functions of KCS genes in pear.

Leaf epicuticular wax as a determinant trait for drought resistance in cereals

The wax coating is an element of the lipid protective layer, the cuticle, which covers the above-ground organs of plants and is the main barrier that prevents non-stomatal water loss. The cuticle helps protect plant surfaces from pathogens and ultraviolet radiation and influences interactions between plants and insects. The accumulation of cuticular wax is one of the mechanisms of adaptation to drought stress. As a result, there are more and more reports on the relationship between cuticular wax and plant resistance to drought. This article deals with the relationship between cuticular waxes and drought resistance in cereals. The paper presents the achievements to date on (i) the relationship between wax biosynthesis and plant response to drought stress using glaucous and nonglaucous near-isogenic lines and on (ii) investigating the role of genes for cuticular wax biosynthesis and transport and the transcription factors that regulate them using mutants with disrupted wax coat formation.

Bacterium-enabled transient gene activation by artificial transcription factors for resolving gene regulation in maize.

Understanding gene regulatory networks is essential to elucidate developmental processes and environmental responses. Here, we studied regulation of a maize (Zea mays) transcription factor gene using designer transcription activator-like effectors (dTALes), which are synthetic Type III TALes of the bacterial genus Xanthomonas and serve as inducers of disease susceptibility gene transcription in host cells. The maize pathogen Xanthomonas vasicola pv. vasculorum was used to introduce 2 independent dTALes into maize cells to induced expression of the gene glossy3 (gl3), which encodes a MYB transcription factor involved in biosynthesis of cuticular wax. RNA-seq analysis of leaf samples identified, in addition to gl3, 146 genes altered in expression by the 2 dTALes. Nine of the 10 genes known to be involved in cuticular wax biosynthesis were upregulated by at least 1 of the 2 dTALes. A gene previously unknown to be associated with gl3, Zm00001d017418, which encodes aldehyde dehydrogenase, was also expressed in a dTALe-dependent manner. A chemically induced mutant and a CRISPR-Cas9 mutant of Zm00001d017418 both exhibited glossy leaf phenotypes, indicating that Zm00001d017418 is involved in biosynthesis of cuticular waxes. Bacterial protein delivery of dTALes proved to be a straightforward and practical approach for the analysis and discovery of pathway-specific genes in maize.

Genome-Wide Identification of the U-Box E3 Ubiquitin Ligase Gene Family in Cabbage (Brassica oleracea var. capitata) and Its Expression Analysis in Response to Cold Stress and Pathogen Infection.

Plant U-box E3 ubiquitin ligases (PUBs) play an important role in growth, development, and stress responses in many species. However, the characteristics of U-box E3 ubiquitin ligase genes in cabbage (Brassica oleracea var. capitata) are still unclear. Here, we carry out the genome-wide analysis of U-box E3 ubiquitin ligase genes in cabbage and identify 65 Brassica oleracea var. capitata U-box E3 ubiquitin ligase (BoPUB) genes in the cabbage genome. Phylogenetic analysis indicates that all 65 BoPUB genes are grouped into six subfamilies, whose members are relatively conserved in the protein domain and exon-intron structure. Chromosomal localization and synteny analyses show that segmental and tandem duplication events contribute to the expansion of the U-box E3 ubiquitin ligase gene family in cabbage. Protein interaction prediction presents that heterodimerization may occur in BoPUB proteins. In silico promoter analysis and spatio-temporal expression profiling of BoPUB genes reveal their involvement in light response, phytohormone response, and growth and development. Furthermore, we find that BoPUB genes participate in the biosynthesis of cuticular wax and in response to cold stress and pathogenic attack. Our findings provide a deep insight into the U-box E3 ubiquitin ligase gene family in cabbage and lay a foundation for the further functional analysis of BoPUB genes in different biological processes.

Molecular mechanisms involved in fruit cracking: A review.

Several fleshy fruits are highly affected by cracking, a severe physiological disorder that compromises their quality and causes high economical losses to the producers. Cracking can occur due to physiological, genetic or environmental factors and may happen during fruit growth, development and ripening. Moreover, in fleshy fruits, exocarp plays an important role, acting as a mechanical protective barrier, defending against biotic or abiotic factors. Thus, when biochemical properties of the cuticle + epidermis + hypodermis are affected, cracks appear in the fruit skin. The identification of genes involved in development such as cell wall modifications, biosynthesis and transport of cuticular waxes, cuticular membrane deposition and associated transcription factors provides new insights to better understand how fruit cracking is affected by genetic factors. Amongst the major environmental stresses causing cracking are excessive water during fruit development, leading to imbalances in cations such as Ca. This review focus on expression of key genes in these pathways, in their influence in affected fruits and the potential for molecular breeding programs, aiming to develop cultivars more resistant to cracking under adverse environmental conditions.

Gene expression profile during seed development of Bixa orellana accessions varying in bixin pigment.

Diverse morphological, cellular and physiological changes occur during seed maturation in Bixa orellana when the seed tissues form specialized cell glands that produce reddish latex with high bixin amounts. Transcriptomic profiling during seed development in three B. orellana accessions (P12, N4 and N5) with contrasting morphologic characteristics showed enrichment in pathways of triterpenes, sesquiterpenes, and cuticular wax biosynthesis. WGCNA allows groups of all identified genes in six modules the module turquoise, the largest and highly correlated with the bixin content. The high number of genes in this module suggests a diversification of regulatory mechanisms for bixin accumulation with the genes belonging to isoprene, triterpenes and carotene pathways, being more highly correlated with the bixin content. Analysis of key genes of the mevalonate (MVA) and the 2C-methyl-D-erythritol-4-phosphate (MEP) pathways revealed specific activities of orthologs of BoHMGR, BoFFP, BoDXS, and BoHDR. This suggests that isoprenoid production is necessary for compounds included in the reddish latex of developing seeds. The carotenoid-related genes BoPSY2, BoPDS1 and BoZDS displayed a high correlation with bixin production, consistent with the requirement for carotene precursors for apocarotenoid biosynthesis. The BoCCD gene member (BoCCD4-4) and some BoALDH (ALDH2B7.2 and ALDH3I1) and BoMET (BoSABATH1 and BoSABATH8) gene members were highly correlated to bixin in the final seed development stage. This suggested a contributing role for several genes in apocarotenoid production. The results revealed high genetic complexity in the biosynthesis of reddish latex and bixin in specialized seed cell glands in different accessions of B. orellana suggesting gene expression coordination between both metabolite biosynthesis processes.

Suppression of cuticular wax biosynthesis mediated by rice LOV KELCH REPEAT PROTEIN 2 supports a negative role in drought stress tolerance.

Drought tolerance is important for grain crops, including rice (Oryza sativa); for example, rice cultivated under intermittent irrigation produces less methane gas compared to rice grown in anaerobic paddy field conditions, but these plants require greater drought tolerance. Moreover, the roles of rice circadian-clock genes in drought tolerance remain largely unknown. Here, we show that the mutation of LOV KELCH REPEAT PROTEIN 2 (OsLKP2) enhanced drought tolerance by increasing cuticular wax biosynthesis. Among ZEITLUPE family genes, OsLKP2 expression specifically increased under dehydration stress. OsLKP2 knockdown (oslkp2-1) and knockout (oslkp2-2) mutants exhibited enhanced drought tolerance. Cuticular waxes inhibit non-stomatal water loss. Under drought conditions, total wax loads on the leaf surface increased by approximately 10% in oslkp2-1 and oslkp2-2 compared to the wild type, and the transcript levels of cuticular wax biosynthesis genes were upregulated in the oslkp2 mutants. Yeast two-hybrid, bimolecular fluorescence complementation, and coimmunoprecipitation assays revealed that OsLKP2 interacts with GIGANTEA (OsGI) in the nucleus. The osgi mutants also showed enhanced tolerance to drought stress, with a high density of wax crystals on their leaf surface. These results demonstrate that the OsLKP2-OsGI interaction negatively regulates wax accumulation on leaf surfaces, thereby decreasing rice resilience to drought stress.

Apple SUMO E3 ligase MdSIZ1 regulates cuticular wax biosynthesis by SUMOylating transcription factor MdMYB30.

A key function of SUMOylation is the coordinated modification of numerous proteins to optimize plant growth and resistance to environmental stress. Plant cuticular wax is deposited on the surface of primary plant organs to form a barrier that provides protection against changes in terrestrial environments. Many recent studies have examined cuticular wax biosynthetic pathways and regulation. However, whether SUMOylation is involved in the regulation of cuticle wax deposition at the posttranslational level remains unclear. Here, we demonstrate that a small ubiquitin-like modifier (SUMO) E3 ligase, SAP AND MIZ1 DOMAIN CONTAINING LIGASE1 (MdSIZ1), regulates wax accumulation and cuticle permeability in apple (Malus domestica Borkh), SUMO E2 CONJUGATING ENZYME 1(MdSCE1) physically interacts with MdMYB30, a transcription factor involved in the regulation of cuticle wax accumulation. MdSIZ1 mediates the SUMOylation and accumulation of MdMYB30 by inhibiting its degradation through the 26S proteasome pathway. Furthermore, MdMYB30 directly binds to the β-KETOACYL-COA SYNTHASE 1 (MdKCS1) promoter to activate its expression and promote wax biosynthesis. These findings indicate that the MdSIZ1-MdMYB30-MdKCS1 module positively regulates cuticular wax biosynthesis in apples. Overall, the findings of our study provide insights into the regulation pathways involved in cuticular wax biosynthesis.

Roles of very long-chain fatty acids in compound leaf patterning in Medicago truncatula.

Plant cuticles are composed of hydrophobic cuticular waxes and cutin. Very long-chain fatty acids (VLCFAs) are components of epidermal waxes and the plasma membrane and are involved in organ morphogenesis. By screening a barrelclover (Medicago truncatula) mutant population tagged by the transposable element of tobacco (Nicotiana tabacum) cell type1 (Tnt1), we identified two types of mutants with unopened flower phenotypes, named unopened flower1 (uof1) and uof2. Both UOF1 and UOF2 encode enzymes that are involved in the biosynthesis of VLCFAs and cuticular wax. Comparative analysis of the mutants indicated that the mutation in UOF1, but not UOF2, leads to the increased number of leaflets in M. truncatula. UOF1 was specifically expressed in the outermost cell layer (L1) of the shoot apical meristem (SAM) and leaf primordia. The uof1 mutants displayed defects in VLCFA-mediated plasma membrane integrity, resulting in the disordered localization of the PIN-FORMED1 (PIN1) ortholog SMOOTH LEAF MARGIN1 (SLM1) in M. truncatula. Our work demonstrates that the UOF1-mediated biosynthesis of VLCFAs in L1 is critical for compound leaf patterning, which is associated with the polarization of the auxin efflux carrier in M. truncatula.

β-ketoacyl-CoA synthase improves the drought tolerance of root restricted grown grapevines by regulating the cuticular wax biosynthesis

• Twenty-five VvKCS genes were identified in grapevine ( Vitis vinifera L.). • VvKCS12 and VvKCS14 were induced by root restriction and drought stress. • Overexpression of VvKCS12 and VvKCS14 improved wax biosynthesis in Arabidopsis. • Overexpression of VvKCS12 and VvKCS14 enhanced drought tolerance in Arabidopsis. Drought stress limits fruit ripening and quality of grape cultivar, particularly with the use of root restriction. However, the mechanism involved in the molecular response to water deficit concomitant to agriculture adjustment remains poorly understood. In the present study, the regulation of two grape β-ketoacyl-CoA synthase ( KCS ) genes obtained with transcriptome analysis whose expression is significantly up-regulated under root restriction. Twenty-five KCS coding genes were identified and analyzed from grapevine genome. Two VvKCSs ( VvKCS12 and VvKCS14 ) had one major transcript expressed at a significantly higher level and up-regulated by drought stress in grape leaves. Subcellular localization analysis demonstrated that VvKCS12 and VvKCS14 were located on endoplasmic reticulum (ER). Heterologous overexpression of VvKCS12 and VvKCS14 promoted leaves cuticular wax biosynthesis and deposition, and enhanced drought tolerance in Arabidopsis. These findings underscore the roles of VvKCS12 and VvKCS14 in the drought response of grapes due to root restriction and pave the way for advanced study of other different functions.

Comparative profiling of volatile organic compounds associated to temperature sensitive resistance to wheat streak mosaic virus (WSMV) in resistant and susceptible wheat cultivars at normal and elevated temperatures

To investigate changes in wheat resistance to wheat streak mosaic virus (WSMV) at normal and elevated temperatures and their effects on the emission of volatile organic compounds (VOCs), the profile of these compounds in WSMV-resistant (R) and susceptible (S) wheat cultivars were analyzed. VOCs were discovered in leaves of R and S cultivars inoculated to WSMV at 20 °C and 32 °C at 24, 48 and 72 h post inoculation (hpi) by GC. MS–HS–SPME. Many VOCs and related metabolic pathways of WSMV inoculated and control plants responded differently at low/high temperatures in R/S cultivars. Compounds such as 6-methyl-5-hepten-2-one and heptanal, represented the maximum abundance in R plants compared to S plants at earlier times especially, 48 hpi/20 °C. Also, at 32 °C, VOCs including heptanal and 2-pentenal had the highest increase in abundance at a later time. In addition, in R plants, several pathways were up-regulated commonly at 20 °C and 32 °C. Although, at 20 °C, they were activated at an earlier time. At 32 °C, most of these pathways were up-regulated at 72 hpi. The most important pathways correlated to combined stresses included lipoxygenase, cuticular wax biosynthesis, fatty acid and lipid biosynthesis. The pathway of phenyl ethanol biosynthesis was up-regulated only at 32 °C, which probably is specifically related to resistance to heat stress. Up-regulation of the lipoxygenase pathway appears to increase vector attractiveness and tolerance to biotic and abiotic stresses. Based on these results, is suggested that there is a particular synchronized regulation of defense-related multi-dimensional VOCs emitting network that is necessary for the occurrence of wheat defense responses against WSMV and/or heat. This research highlights the challenge of global warming and the breakdown of plant resistance to viruses which requires metabolic engineering of VOCs for sustainable agriculture.

Co-expression of stress-responsive regulatory genes, MuNAC4, MuWRKY3 and MuMYB96 associated with resistant-traits improves drought adaptation in transgenic groundnut (Arachis hypogaea l.) plants.

Groundnut, cultivated under rain-fed conditions is prone to yield losses due to intermittent drought stress. Drought tolerance is a complex phenomenon and multiple gene expression required to maintain the cellular tolerance. Transcription factors (TFs) regulate many functional genes involved in tolerance mechanisms. In this study, three stress-responsive regulatory TFs cloned from horse gram, (Macrotyloma uniflorum (Lam) Verdc.), MuMYB96, involved in cuticular wax biosynthesis; MuWRKY3, associated with anti-oxidant defense mechanism and MuNAC4, tangled with lateral root development were simultaneously expressed to enhance drought stress resistance in groundnut (Arachis hypogaea L.). The multigene transgenic groundnut lines showed reduced ROS production, membrane damage, and increased superoxide dismutase (SOD) and ascorbate peroxidase (APX) enzyme activity, evidencing improved antioxidative defense mechanisms under drought stress. Multigene transgenic plants showed lower proline content, increased soluble sugars, epicuticular wax content and higher relative water content suggesting higher maintenance of tissue water status compared to wildype and mock plants. The scanning electron microscopy (SEM) analysis showed a substantial increase in deposition of cuticular waxes and variation in stomatal number in multigene transgenic lines compared to wild type and mock plants. The multigene transgenic plants showed increased growth of lateral roots, chlorophyll content, and stay-green nature in drought stress compared to wild type and mock plants. Expression analysis of transgenes, MuMYB96, MuWRKY3, and MuNAC4 and their downstream target genes, KCS6, KCR1, APX3, CSD1, LBD16 and DBP using qRT-PCR showed a two- to four-fold increase in transcript levels in multigene transgenic groundnut plants over wild type and mock plants under drought stress. Our study demonstrate that introducing multiple genes with simultaneous expression of genes is a viable option to improve stress tolerance and productivity under drought stress.

Characterization of Glossy Spike Mutants and Identification of Candidate Genes Regulating Cuticular Wax Synthesis in Barley (Hordeum vulgare L.).

Cuticular waxes comprise the hydrophobic layer that protects crops against nonstomatal water loss and biotic and abiotic stresses. Expanding on our current knowledge of the genes that are involved in cuticular wax biosynthesis and regulation plays an important role in dissecting the processes of cuticular wax metabolism. In this study, we identified the Cer-GN1 barley (Hordeum vulgare L.) mutant that is generated by ethyl methanesulfonate mutagenesis with a glossy spike phenotype that is controlled by a single recessive nuclear gene. A physiological analysis showed that the total cuticular wax loads of Cer-GN1 were one-third that of the progenitor wild-type (WT), and its water loss rate was significantly accelerated (p < 0.05). In addition, Cer-GN1 was defective in the glume's cuticle according to the toluidine blue dye test, and it was deficient in the tubule-shaped crystals which were observed on the glume surfaces by scanning electron microscopy. Using metabolomics and transcriptomics, we investigated the impacts of cuticular wax composition and waxy regulatory genes on the loss of the glaucous wax in the spikes of Cer-GN1. Among the differential metabolites, we found that 16-hydroxyhexadecanoic acid, which is one of the predominant C16 and C18 fatty acid-derived cutin monomers, was significantly downregulated in Cer-GN1 when it was compared to that of WT. We identified two novel genes that are located on chromosome 4H and are downregulated in Cer-GN1 (HvMSTRG.29184 and HvMSTRG.29185) that encode long-chain fatty acid omega-monooxygenase CYP704B1, which regulates the conversion of C16 palmitic acid to 16-hydroxyhexadecanoic acid. A quantitative real-time PCR revealed that the expression levels of HvMSTRG.29184 and HvMSTRG.29185 were downregulated at 1, 4, 8, 12, and 16 days after the heading stage in Cer-GN1 when it was compared to those of WT. These results suggested that HvMSTRG.29184 and HvMSTRG.29185 have CYP704B1 activity, which could regulate the conversion of C16 palmitic acid to 16-hydroxyhexadecanoic acid in barley. Their downregulation in Cer-GN1 reduced the synthesis of the cuticular wax components and ultimately caused the loss of the glaucous wax in the spikes. It is necessary to verify whether HvMSTRG.29184 and HvMSTRG.29185 truly encode a CYP704B1 that regulates the conversion of C16 palmitic acid to 16-hydroxyhexadecanoic acid in barley.

Nitric oxide effects on postharvest and Alternaria-infected pear fruit

Postharvest quality deterioration and pathogenic infestation interfere with the development of the pear industry. Nitric oxide (NO), as an emerging potential postharvest fruit protectant, can simultaneously stop fruit senescence and improve disease resistance. Global visualization of NO in the pears postharvest process is necessary. In this study, NO gas was used to fumigate 'Korla' fragrant pear, we explored changes in phenotypic indicators and gene expression of pear fruit during 120 d of refrigeration and 15 d of Alternaria sp. infection. Similar to 1-MCP treatment, NO treatment hindered the deterioration of pear quality, reduced and delayed the respiration and ethylene release rates, increased the contents of major quality traits and nutrients, and downregulated many carbohydrate metabolism genes. NO reduced Alternaria sp. damage, activated the activity of resistance enzymes of infected pears, and increased the contents of antioxidant substances. A large number of induced systemic resistance genes were upregulated in infected pears. Pear cuticular wax biosynthesis responded to NO treatment with increased total wax content and upregulation of the core genes ECERIFERUM (CER) and 3-KETOACYL-COA SYNTHASE (KCS). These results provide guidance for the application of NO in fruit postharvest.

BrWAX3, Encoding a β-ketoacyl-CoA Synthase, Plays an Essential Role in Cuticular Wax Biosynthesis in Chinese Cabbage.

In this study, we identified a novel glossy mutant from Chinese cabbage, named SD369, and all wax monomers longer than 26 carbons were significantly decreased. Inheritance analysis revealed that the glossy trait of SD369 was controlled by a single recessive locus, BrWAX3. We fine-mapped the BrWAX3 locus to an interval of 161.82 kb on chromosome A09. According to the annotated genome of Brassica rapa, Bra024749 (BrCER60.A09), encoding a β-ketoacyl-CoA synthase, was identified as the candidate gene. Expression analysis showed that BrCER60.A09 was significantly downregulated in all aerial organs of glossy plants. Subcellular localization indicated that the BrCER60.A09 protein functions in the endoplasmic reticulum. A 5567-bp insertion was identified in exon 1 of BrCER60.A09 in SD369, which lead to a premature stop codon, thus causing a loss of function of the BrCER60.A09 enzyme. Moreover, comparative transcriptome analysis revealed that the ‘cutin, suberine, and wax biosynthesis’ pathway was significantly enriched, and genes involved in this pathway were almost upregulated in glossy plants. Further, two functional markers, BrWAX3-InDel and BrWAX3-KASP1, were developed and validated. Overall, these results provide a new information for the cuticular wax biosynthesis and provide applicable markers for marker-assisted selection (MAS)-based breeding of Brassica rapa.

Functional conservation of an AP2/ERF transcription factor in cuticle formation suggests an important role in the terrestrialization of early land plants.

The formation of a hydrophobic cuticle layer on aerial plant parts was a critical innovation for protection from the terrestrial environment during the evolution of land plants. However, little is known about the molecular mechanisms underlying cuticle biogenesis in early terrestrial plants. Here, we report an APETALA2/Ethylene Response Factor (AP2/ERF) transcriptional activator, PpWIN1, involved in cutin and cuticular wax biosynthesis in Physcomitrium patens and Arabidopsis. The transcript levels of PpWIN1 were 2.5-fold higher in gametophores than in the protonema, and increased by approximately 3- to 4.7-fold in the protonema and gametophores under salt and osmotic stresses. PpWIN1 harbouring transcriptional activation activity is localized in the nucleus of tobacco leaf epidermal cells. Δppwin1 knockout mutants displayed a permeable cuticle, increased water loss, and cutin- and wax-deficient phenotypes. In contrast, increased total cutin and wax loads, and decreased water loss rates were observed in PpWIN1-overexpressing Arabidopsis plants. The transcript levels of genes involved in cutin or wax biosynthesis were significantly up-regulated in PpWIN1-overexpressing Arabidopsis lines, indicating that PpWIN1 acts as a transcriptional activator in cuticle biosynthesis. This study suggests that Arabidopsis WIN1/SHN1 orthologs may be functionally conserved from early to vascular land plants.

Mutation of OsMYB60 reduces rice resilience to drought stress by attenuating cuticular wax biosynthesis.

The cuticular wax layer on leaf surfaces limits non-stomatal water loss to the atmosphere and protects against pathogen invasion. Although many genes associated with wax biosynthesis and wax transport in plants have been identified, their regulatory mechanisms remain largely unknown. Here, we show that the MYB transcription factor OsMYB60 positively regulates cuticular wax biosynthesis and this helps rice (Oryza sativa) plants tolerate drought stress. Compared with the wild type (japonica cultivar 'Dongjin'), osmyb60 null mutants (osmyb60-1 and osmyb60-2) exhibited increased drought sensitivity, with more chlorophyll leaching and higher rates of water loss. Quantitative reverse-transcription PCR showed that the loss of function of OsMYB60 led to downregulation of wax biosynthesis genes, leading to reduced amounts of total wax components on leaf surfaces under normal conditions. Yeast one-hybrid, luciferase transient transcriptional activity, and chromatin immunoprecipitation assays revealed that OsMYB60 directly binds to the promoter of OsCER1 (a key gene involved in very-long-chain alkane biosynthesis) and upregulates its expression. Taken together, these results demonstrate that OsMYB60 enhances rice resilience to drought stress by promoting cuticular wax biosynthesis on leaf surfaces.